|Publication number||US7390653 B2|
|Application number||US 10/725,607|
|Publication date||Jun 24, 2008|
|Filing date||Dec 2, 2003|
|Priority date||Dec 4, 2002|
|Also published as||EP1572983A1, EP1572983A4, US20040110273, WO2004050864A1|
|Publication number||10725607, 725607, US 7390653 B2, US 7390653B2, US-B2-7390653, US7390653 B2, US7390653B2|
|Inventors||Roger Akers, William J. Anderson, Adrian F. Dinges, Jr., Stephen S. Navran, Jr.|
|Original Assignee||Synthecon, Inc.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (18), Non-Patent Citations (4), Referenced by (6), Classifications (38), Legal Events (4)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application claims priority to U.S. Patent Application Ser. No. 60/430,795 filed Dec. 4, 2002 by inventors Roger Akers, William Anderson, Steve Navran and Adrian Dinges and entitled “Culture Vessels for Biologicals.” The entire text of the above-referenced disclosure is incorporated by reference herein.
1. Field of the Invention
The present invention relates to a culture chamber for culturing cells, cellular aggregates, particles, tissues and organoids. More particularly, the present invention relates to a culture chamber having one or more molecular weight cut-off membranes transversing the chamber, wherein incoming media enters the chamber through an inlet and then passes through a membrane into the culture chamber and the exiting media passes through the membrane and then out the chamber outlet.
2. Description of the Related Art
The biomanufacturing industry is experiencing rapid growth. One aspect of that growth is that the demand for the production of new therapeutic protein products is greatly exceeding capacity. To keep pace with the drugs and antibodies generated in cell culture that are coming to market, biomanufacturing industry requirements for therapeutic protein production is expected to increase yet another five or six fold over the next couple of years. Since the industry is already exceeding current capacity for the production of therapeutic proteins, new processes and apparatuses are needed that are more efficient and that can be scaled up for the production of larger quantities of therapeutic proteins.
One of the major problems in producing therapeutic protein products is the time and costs of purifying the desired protein from the cell media. It is estimated that two thirds of the time and costs of manufacturing proteins from cell cultures is related to the separation of the desired protein product from the waste products. Thus, there is a need to produce a more concentrated protein product upstream to reduce the downstream processing required to purify the protein product.
In addition, the expense of producing biologicals in aseptic bioreactors is exacerbated by the required cleaning, sterilization and validation of the standard stainless steel or glass bioreactors by the customer. There is a continuing need to develop lightweight, presterilized, disposable culture chambers with simple connections to existing equipment that require little training to operate, yet provide the necessary gas transfer and nutrient mixing required for successful cell cultures. If the culture chambers used in producing therapeutic proteins could be made disposable, a reduction in the risk of cross contamination, the time and expense in changing from the production of one protein to another, and the downtime needed for equipment changeover between production runs would be realized.
The invention contemplates a culture chamber having a fluid-filled compartment in which cells, tissues and other biologicals are cultured. The culture chamber is transversed by one or more molecular weight cut-off membranes. Incoming nutrients are transported through the membrane into the culture chamber and metabolic waste products are transported away from the fluid-filled culture compartment through the membrane and out the chamber outlet.
One aspect of the present invention is a culture chamber comprising: a tubular housing; a growth compartment within the housing; a fluid inlet; a fluid outlet; and a membrane carrier assembly transversing the growth compartment including a support cylinder having a first end in communication with the fluid inlet and a second end in communication with the fluid outlet, a molecular weight cut-off membrane secured to an exterior surface of the support cylinder, and a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the fluid inlet and the fluid outlet.
Another aspect of the present invention is a culture vessel comprising: (a) a housing having a right circular cylindrical sleeve having a first and a second end; and a first and a second end fitting including an interior projection, the interior projection having an outer diameter that sealingly fits within a bore of the sleeve to seal the first and second ends of the sleeve, a nozzle on an exterior side of the end fitting, a counterbore in the interior projection, and a through bore passing through the end fitting, the through bore extending from the nozzle to the counterbore; (b) a growth compartment within the bore of the sleeve; (c) a support cylinder transversing the growth compartment, the support cylinder having a first and second end, each end having a fluid channel extending from the end of the cylinder to an exterior surface of the cylinder in a mid-section of the cylinder, wherein the fluid channel is in communication with the through bore of the end fitting whenever the cylinder is positioned in the counterbore of the interior projection; and (d) a molecular weight cut-off membrane secured to an exterior surface of the support cylinder to provide a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the fluid channels of the support cylinder.
Another aspect of the present invention is a disposable culture bag comprising: (a) a flexible outer wall having a first end, a second end, an internal side, and an external side, wherein the internal side of the wall is positioned to face an interior of the culture bag; (b) an inlet means fused to the first end of the wall, wherein the inlet means includes an inlet end piece having an inlet interior counterbore and an inlet through bore; (c) an outlet means fused to the second end of the wall, wherein the outlet means includes an outlet end piece having an outlet interior counterbore and an outlet through bore; and (d) a membrane carrier assembly connecting the inlet through bore to the outlet through bore including a support cylinder positioned at a first end in the inlet interior counterbore and at a second end in the outlet interior counterbore, the support cylinder having a fluid channel at the first end and second ends in communication with the inlet and outlet through bores, a molecular weight cut-off membrane secured to an exterior surface of the support cylinder, and a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the inlet and outlet through bores.
Yet another aspect of the present invention is a method of culturing cells in a culture chamber having a growth compartment transversed by a support cylinder surrounded by a molecular weight cut-off membrane to form a fluid chamber between the support cylinder and the membrane, the fluid chamber in fluid communication with a fluid inlet and a fluid outlet, the method comprising the steps of: (a) filling the growth compartment with a nutrient media; (b) placing a cell culture mixture in the nutrient media in the growth compartment; (c) rotating the culture chamber; (d) pumping additional nutrient media into the fluid inlet, through the fluid chamber, and out the fluid outlet; and (e) transporting a number of compounds having a molecular weight less than the molecular weight cut-off of the membrane across the membrane.
The foregoing has outlined several aspects of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and the specific embodiments disclosed might be readily utilized as a basis for modifying or redesigning the method or process for carrying out the same purposes as the invention. It should be realized that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims.
For a more complete understanding of the present invention, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:
The culture chamber of the present invention has a culture compartment in which cells, tissues and other biologicals are cultured in fluid media. The culture compartment is transversed by one or more molecular weight cut-off membranes attached to a membrane carrier assembly. Incoming nutrients and/or biological modifiers are transported through the membrane into the culture compartment and metabolic waste products are transported away from the fluid-filled culture compartment through the membrane and out the chamber outlet. Both reusable and disposable culture chambers are described for culturing cells, cell aggregates, particles, tissues and organoids.
The molecular weight cut-off membranes that transverse the culture compartment permit two-way perfusion into and out of the cell growth chamber. The membranes allow nutrients, growth factors and gases in the input fluid entering through the inlet means of the culture chamber to perfuse into and throughout the interior of the culture compartment. At the same time waste products are able to perfuse out of the chamber through the molecular weight cut-off membrane in the reverse direction into the exiting fluid that passes through the interior of the membrane. The use of multiple membranes transversing the culture compartment ensures that fluid entering through the inlet means of the culture chamber perfuses throughout the interior of the culture compartment and is well mixed with the media within the culture compartment. The culture chambers of the present invention include both reusable chambers and chambers having a reusable housing containing a disposable bioreactor bag.
Referring now to the drawings, and initially to
Reusable Culture Chambers
The biologicals being cultured in the rotatable chamber require nutrients, so fluid-conducting swivels 50, stopcock valves 60, and fluid inlet tubing 72 and outlet tubing 71 are provided on the ends of the housing 10 so that the entry and exit of media is controlled. Multiple radial wall penetration ports 25 are provided in the annular wall of sleeve 24 to allow the introduction of one or more fill ports 40 and one or more vent ports 44. The swivel 50, the fill port 40, and the vent port 44 are described in more detail below.
The particulars of the construction of the components of culture chamber 10 are best understood with reference to
The right circular cylindrical sleeve 24 can be made of a variety of materials such as glass, stainless steel or plastic. Preferably the reusable cell culture chamber is constructed of plastic, typically a transparent plastic such as an acrylic plastic for the cylindrical sleeve 24 and opaque plastics such as Kynar™ or Delrin™ for the other rigid pieces such as the end pieces 11. Suitable plastics have substantially zero porosity and are impermeable to gases and non-reactive to biological media and its components. Suitable construction materials must also be able to undergo multiple sterilizations by steam, gas, or radiation without deforming, cracking or otherwise being rendered unusable.
Although not shown in
The growth compartment 5 is located between the interior bore of the sleeve 24, the membrane carrier assembly 27, and the interior ends of the interior projection 15 of the end pieces 11. The growth compartment 5 is transversed by a membrane carrier assembly 27 as shown in
Centrally deployed with a close fit around the exterior of cylinder 28 is a tubular molecular weight cut-off membrane 37. Membrane 37 is flexible with a limited amount of elastic stretch capability. The construction of membrane 37 is very carefully controlled so that the number of molecules, having a molecular weight in excess of the specific limiting molecular weight cut-off value of the membrane 37, transfused through the membrane in either direction is statistically very small and rapidly decreases as a function of increasing molecular weight. Thus, there is essentially no passage of molecules, much larger than the molecular weight cut-off value of the membrane, through the membrane 37. The molecular weight cut-off value of the membrane 37 is preselected so that nutrients and growth factors, as well as metabolic waste products, can easily transfuse through the membrane, whereas larger cellular products can be retained. For example, Factor VIII (having a molecular weight of about 350,000 daltons) or IgG monoclonal antibodies (having a molecular weight of about 155,000 daltons) produced by genetically engineered bacteria or cells can be retained by a membrane with a molecular weight cut-off value of about 100,000 daltons; whereas the majority of serum albumin (having a molecular weight of about 67,500 daltons and making up 55% to 62% of serum protein) would be allowed to pass through the membrane.
Currently bioreactors and culture chambers are designed to have a filter to keep the cells within the chamber and to allow the desired therapeutic protein to pass out of the chamber with the waste products. The present invention also allows the user to select a membrane having a molecular weight cut-off value that would allow the desired protein to pass out of the culture chamber with the waste products. However, the present invention also permits the user to select a membrane having a smaller molecular weight cut-off value than the desired protein, so that the desired protein is retained within the culture chamber and is concentrated with time as the cells multiply and continue to produce the desired protein. Since the culture chamber 10 is reusable, the membrane carrier assembly 27 can be assembled with membranes 37 having a variety of molecular weight cut-off values depending on the protein being produced.
Furthermore, the membrane carrier assembly 27 of the present invention provides a media circulating system that allows the user to constantly monitor certain media parameters in the culture chamber (e.g., the pH) and to adjust those parameters by adjusting the media being pumped into the culture chamber, thereby maintaining optimum conditions for the cells being cultured.
As shown in
As can be seen in
The construction details of the culture chamber 100 are best understood by reference to
Reduced diameter coaxial right circular cylindrical interior projection 115 extends inwardly on the transverse face of the interior end of the central section of end piece 111. The exterior cylindrical surface of interior projection 115 has, in order from the interior transverse face of end piece 111, first and second annular male O-ring grooves 117 and 116. Elastomeric O-ring 118 is mounted in O-ring groove 117. At the interior end of interior projection 115 of end piece 111, an array of flat-bottomed counterbores 119 (in this case four) are all located at the same radius and have coaxial blind holes 120 at their inner ends. Each blind hole 120 intersects the outer end of a corresponding radial hole 121. Although not shown, an optional lead-in chamfer may be provided at the mouth of counterbore 119 in order to facilitate the stabbing of an O-ring seal.
Right circular cylindrical sleeve 124, preferably made of acrylic plastic as previously described for sleeve 24, may be provided with lead-in tapers on its interior corners to facilitate the stabbing of O-rings. The interior bore of sleeve 124 is a close sliding fit to the outer diameter of interior projection 115 of end fitting 111, thereby permitting O-ring 118 to sealingly engage the bore of sleeve 124. As stated previously, the sleeve 124 has multiple radial wall penetration ports 125 to allow the mounting of fill ports 40 and vent ports 44 (not shown, but similar to those in the culture chamber 10). The fill ports 40 are used for inserting fluids into or removing fluids from the interior of the growth compartment 105 while the chamber 100 is in service but not rotating. Vent ports 44 are used to allow gas to escape from the growth compartment 105 as it is being filled with media. Each end of sleeve 124 is stabbed over interior projection 115, sealed against an O-ring 118, and shouldered against the interior transverse face of the central portion of an end piece 111, so that a sealed growth compartment 105 is formed between end pieces 111, the interior of tube 124, and around the membrane carrier assemblies 27.
The corresponding counterbores 119 in both end pieces 111 are coaxially aligned so that a cylindrical membrane carrier assembly 27 is stabbed and bottomed in the corresponding counterbores of each of the two end pieces 111. The membrane carrier assemblies 27 are identical to that used in the culture chamber 10 and shown in
A third embodiment, culture chamber 200, of a reusable culture chamber of the present invention is shown in
The culture chamber 200 is typically round in construction as it is designed to be supported on and rotated by a roller drive that rotates the chamber about its axis. Fluid-conducting swivels 50, stopcock valves 60, and fluid inlet tubing 72 and outlet tubing 71 are provided on the ends of the housing 200 so that the nutrient fluid is added to and mixed with the media in the growth compartment 205 and waste products are removed from the growth compartment 205 through the membrane and out the culture chamber 200. Multiple radial wall penetration ports 125 are provided in the annular wall of sleeve 124 to allow the introduction of one or more fill ports 40 and one or more vent ports 44.
As seen in
Outlet end piece 239 is conformed similarly to the inlet end piece 111, but is built in two main pieces, inner plug 240 and outer flange 250, in order to permit assembly. Inner plug 240 is a right cylindrical disk having the same inner diameter as the sleeve 124 with which it is mated. Inner plug 240 has a first O-ring groove 241 and a second O-ring groove 242 on its outer circumference. O-ring 243, which seals between sleeve 124 and inner plug 240, is positioned in the male O-ring groove 242.
On its inner transverse face, plug 240 has a number of equispaced flat-bottomed counterbored socket holes 245. For example,
Outer flange 250 is a right circular cylindrical disk with its outer diameter equal to that of sleeve 124 and having a concentric reduced diameter right circular cylindrical extension 255 on its outer transverse face. Coaxial push-on hose barb attachment neck 258 is positioned on the outer transverse face of extension 255 and axial through hole 257 penetrates through both the attachment neck 258 and the rest of the body of outer flange 250. The inner transverse face of outer flange 250 abuts the transverse end of sleeve 124 and has concentric O-ring face groove 251 mounting O-ring 252 located there. O-ring 252 seals between inner plug 240 and outer flange 250 to prevent leakage in the radial direction.
Shallow flat-bottomed counterbore 256 is located on the interior transverse face of flange 250 inside of O-ring groove 251 and is intersected by through hole 257. The diameter of counterbore 256 is such that it extends to the outer periphery of the through holes 246 in inner plug 240 so that fluid communication is possible. Four through clearance holes 254 penetrate outer flange 250 on the same bolt circle pattern as for drilled and tapped holes 244 on the inner plug 240. Machine screws 260 pass through holes 254 and are threadedly engaged into tapped holes 244 in order to clamp inner plug 240 and outer flange 250 together to form outlet end piece 239.
End plug 270 is a right circular cylinder with concentric through bore 271 extending through its length. The outer end of bore 271 is drilled and tapped for assembly purposes so that it may be blindly aligned and pulled into engagement with its comating mounting counterbored socket 245 in inner plug 240. The exterior of end plug 270 has, from its outer end, a lead-in taper to facilitate stabbing into a mounting receptacle such as socket 245 or 119 in end piece 111, male O-ring groove 273, and rectangular profile retention groove 272. O-ring 274 is mounted in groove 273.
The tubular molecular weight cut-off membrane 37 used in culture chamber 200 is identical in its shape and size to that used in the other configurations of the culture chamber. Membrane 37 is stretched over the interior end of end plug 270 past groove 273 and then sealingly retained on plug 270 by using O-ring 32 to force the membrane tightly into groove 272. One end plug 270 with its O-rings 274 and 32 is used on each end of the membrane 37 to complete the assembly of the diffuser assembly 227.
To construct cell culture chamber 200, each assembled diffuser assembly 227 has one end inserted sealingly into its corresponding socket 119 in end piece 111 until it bottoms out. The sleeve 124 is then assembled over the cylindrical inner end 115 of end piece 111 with O-ring 118 in place to effect a seal. At this point, inner plug 240 is aligned with end piece 111, assembled with its O-ring 243, and machine screws or studs (not shown) are extended inwardly through holes 246 and engaged in the tapped portion of bores 271 of the free end of diffuser assembly 227. After inner plug 240 is installed with its outer transverse face approximately flush with the end of sleeve 124, the screws or studs inserted into bores 271 of the diffuser assembly 227 can be used to pull the individual engaged end plugs 270 fully into the sockets 245 of inner plug 240 where they are in sealing engagement. At that point, the assembly screws or studs can be removed from plug 240 and end plugs 270 so that the end flange 250 can be attached to plug 240 using screws 260 and the assembly completed.
Culture Chambers with Disposable Bioreactor Bags
The present invention also includes culture chambers having a reusable bag housing to provide support for a disposable bioreactor bag. Since the bag assemblies are designed to be supported by a bag support housing, very large capacity bag assemblies can be used to scale-up production procedures. The bag assemblies described herein are made to hold anywhere from milliliters to thousands of liters of fluid. Furthermore, the bag assemblies described herein are pre-sterilized before use. Sterilization is typically done using gamma radiation or gas.
The bioreactor bags are housed in a rotatable housing and rotated by a drive mechanism as described in copending U.S. patent application Ser. No. 10/283,000 entitled “Disposable Culture Bag” and filed on Oct. 29, 2002. U.S. patent application Ser. No. 10/283,000 is incorporated herein by reference. The disposable bioreactor bags have rigid end pieces that can be configure to carry one or more membrane carrier assemblies 27 or one or more diffuser assemblies 227 with molecular weight cut-off membranes 37.
All of the components of bag assembly 301 which contact the biological media and which are supplied to and removed from the bag assembly are biologically non-reactive, non-toxic and exhibit low protein binding properties. Bag assembly 301 and its constituent components are fused together with heat and pressure. However, the term fusing will be used with reference to joining elements of the bag assemblies of this invention where the elements are joined using heat, adhesive, ultrasonic welding, or other suitable means to effect the connections.
Preferably the bag 320 is made of a plurality of layers such as the multilayered fabric construction used in the synthesis of custom bags manufactured by Newport Biosystems, Inc. (Anderson, Calif.). For example, a typical four-ply fabric construction would have individual layers, sequentially from the outer bag layer, of nylon, polyvinyldichloride (PVDC), a linear low density polyethylene (LLDPE), and a LLDPE inner layer for contacting the cells and the biological media. The plies of the bag have thicknesses with physical and molecular properties to provide the desired puncture strength, tensile strength, flexural strength, cell and gas and liquid permeabilities in appropriate ranges, and weldability and/or bondability or fusibility. The desired permeabilities typically are low or zero. However, variations of the bag 320 are designed to be gas-permeable for certain applications.
Bag end 305 is a right circular cylindrical disk having a concentric connecting neck 306 on its outer transverse face. Connecting neck 306 has, in sequential order from the outer face of bag end 305, a straight cylindrical shank 307, an externally projecting frustro-conical latching shoulder 308, and a second straight cylindrical outer shank 309. The inward end of the frustro-conical latching shoulder 308 is a transverse shoulder, while the outward end has the same diameter as that of second shank 309. The interior end of bag end 305 has a concentric flat-bottomed counterbore 310 which is intercepted by axial through hole 311 which penetrates through the remainder of bag end 305 and connecting neck 306. The diameter of counterbore 310 is such that it provides a close fit to the outer diameter of support cylinder 28 of the membrane carrier assembly 27. Bag end 305 is circumferentially welded or otherwise fused on its outer diameter to the end of bag 320.
The membrane carrier assembly 27 is substantially identical to that used in culture chambers 10 and 100 and shown in detail in
The disposable bioreactor bag assemblies have flexible walls and are supported by a bag support housing. Numerous configurations of a bag support housing can be used. One example of a bag support housing 330, shown in
The bag support end 331 has a thin-walled right circular cylindrical section and a transverse bulkhead 333 at a first end of the cylindrical section. The inner diameter of the bag support end 331 is sized to provide a close slip fit to the filled bioreactor bag assembly 301. The transverse bulkhead has a concentric circular hole 334 sized to closely fit with the exterior cylindrical surface of bag end 305 and a thickness of approximately 0.5 to 0.1 inch. The bag support end 331 has female threads 332 at its second, opposed end. Multiple radial wall penetration ports 436 are provided in the annular wall of the bag support end 331 to allow the introduction of one or more vent ports 425 and one or more fill ports 423 in the bag assembly 301.
Bag support closure 338 is a round disk with a central circular hole 339 that closely fits the outer diameter of bag end 305 and outer holes designed to fit vent ports 425 or fill ports 423 in the bas assembly 301. The bag support closure 338 has a thickness of approximately 0.5 to 0.1 inch. The exterior cylindrical surface of bag support closure has male threads 340 comatable with the female threads 332 of bag support end 331 so that the two items may be screwed together to create a housing 330 having coaxial end holes 334 and 339.
Drive assembly 360 may have a variety of designs. In this embodiment the drive assembly 360 is similar to that used in U.S. Pat. No. 6,080,581, which is hereby incorporated by reference, where a rigid cylinder was used as the rotating bioreactor. Two parallel, spaced-apart, journaled shaft assemblies support the bag support housing 330 that contains bag assembly 301. Each of the two journaled shaft assemblies has a set of one or more equidiameter rollers 375 mounted on it and the bag support housing 330 is tangential to both sets of rollers. At least one and, possibly, both shaft assemblies are driven. A drive assembly 360 consists of variable speed motor 362 which has its output shaft 363 connected to main shaft 368 by cylindrical shaft coupling 364. Main shaft 368 is journaled in two places near its ends by bearings 369, which are in turn supported by pillow blocks 370. Equidiameter rollers 375 are concentrically mounted on main shaft 368 adjacent the pillow blocks 370 and positioned to support the bag support housing 330 close to its ends.
Motor 362 is mounted on base plate 361, as are the pillow blocks 370. If an undriven idler shaft assembly is used, then the shaft coupling 364 and motor 362 are omitted. If both shaft assemblies are driven, then they must be synchronized to run at the same speed. The motor driver or drivers are not indicated in
Bag assembly 401, like the bag assembly 301, includes multiple service ports such as a gas venting port 425 and a fill port 423. The fill port 423 is bonded into a penetration 424 in the side of bag 420. The vent port 425 is bonded into a penetration 426 in the side of bag 420. The attachments of ports 423 and 425 into bag 420 are reinforced by sealing grommets on both sides of the bag wall, as is well understood by those skilled in the art.
As described for the bag assembly 301, the bag assembly 401 has its constituent components welded or bonded or fused together. Furthermore, all of the components of the bag assembly 401 contacting the biological media are biologically non-reactive, non-toxic and exhibit low protein binding properties.
Preferably the bag 420 is made of a plurality of layers such as the multilayered fabric construction used in the synthesis of custom bags manufactured by Newport Biosystems, Inc. (Anderson, Calif.), as previously described for the bag 320 in the fourth cell growth chamber embodiment 300.
Bag end 405 is a right circular cylindrical disk having a concentric connecting neck 406 on its outer transverse face. Connecting neck 406 has, in sequential order from the outer face of bag end 405, a straight cylindrical shank 407, an externally projecting frustro-conical latching shoulder 408, and a second straight cylindrical outer shank 409. The inward end of the frustro-conical latching shoulder 408 is a transverse shoulder, while the outward end has the same diameter as that of second shank 409. Coaxial hole 411 is located on the axis of connecting neck 406 and penetrates through the length of connecting neck 406 and part way into the main cylindrical body of bag end 405.
Hole 411 is intersected by four equispaced radial cross holes 412 which extend from the central axis of bag end 405 approximately halfway to the outer cylindrical face. The interior end of bag end 405 has four equispaced flat-bottomed counterbores 414. Each counterbore 414 is intercepted by a short coaxial blind hole 413, which is in turn intercepted by a cross hole 412. Accordingly, a fluid path is created from the hole 411 transversing connecting neck 406, through the cross holes 412 and the short connecting holes 413 into each of the counterbores 414.
The diameter of counterbore 414 is such that it provides a close fit to the outer diameter of support cylinder 28 of the membrane carrier assembly 27. A bag end 405 is circumferentially welded or otherwise fused on its outer cylindrical face to the corresponding end of bag 420 at its end opening. The bag ends 405 are aligned so that their counterbores 414 are coaxial prior to attachment to the bag 420.
The membrane carrier assembly 27 transversing the bag assembly 401 has been previously described and is shown in detail in
Bag assembly 401 has identical inlet and outlet fluid coupling swivel joints 350 to allow the. bag assembly 401 to freely rotate with its bag support housing 430 while connected to the input and output fluid feed tubings 72 and 71. The swivel 350 is latched onto neck 406 so that relative axial motion is prevented by having the shoulder of frustro-conical latching shoulder 408 abut the corresponding shoulder of the recess 356 of counterbore 353 in the swivel 350. The connection is a one-way snap connection effected when neck 406 is forced into counterbore 353.
Although the bag support housing 430 may be made any shape, the bag support housing is preferably a circular cylinder to ease its horizontal rotation. Bag support assembly 430, as shown in detail in
Central concentric bores 433 in both the transverse inlet and outlet bulkhead ends of the bag support assembly 430 are designed to fit around the bag ends 405, while additional bores 434 are located on the diametrical split so that they can readily accommodate insertion of fill ports 423 into the split bore. One or more additional radial ports 435 with clearance to permit insertion of a corresponding number of gas removal ports 425 are appropriately positioned in the cylindrical walls of the upper 431 and lower 432 portions of the bag support housing 430. The positioning of the radial ports 435 need not be on the diametrical split of the bag support assembly 430.
One advantage of bag support housing 430 is that a bag assembly 401 made with an inlet and outlet system incorporated into the bag assembly 401 can be inserted into the bag support housing 430 without having to disconnect any tubing or connectors. The bag support housing 430 may be constructed of a variety of materials known in the art, but will preferably be constructed of either a metal, such as stainless steel, or a solid plastic, such as Plexiglas™ or acrylic. A drive assembly, such as drive assembly 360 described above, horizontally rotates the bag support assembly 430.
A third embodiment of the culture chamber 500 adapted for use with a disposable bioreactor bag 501 is shown in
As seen in
End plug 530 is a right circular cylinder with concentric through hole 571 extending through its length. The exterior of end plug 530 has, from its outer end, male O-ring groove 521, and rectangular profile retention groove 523. O-ring 522 is mounted in groove 521.
The tubular molecular weight cut-off membrane 37 used in culture chamber 500 is identical in its shape and size to that used in the other configurations of the culture chamber. Membrane 37 is stretched over the interior end of end plug 530 past groove 522 and then sealingly retained on plug 530 by using O-ring 32 to force the membrane tightly into groove 523. One end plug 270 with its O-rings 522 and 32 is used on each end of the membrane 37 to complete the assembly of the diffuser assembly 557.
To construct cell culture chamber 500, the assembled diffuser assembly 557 has one end inserted sealingly into its corresponding flat-bottomed counterbore 310 in end piece 305 until it bottoms out. The other end of the diffuser assembly 557 is then aligned with the other end piece 305 and the end plug 530 inserted into its corresponding counterbore 310 until it bottoms out. The sides of the bag 520 are then fused to the two end pieces 305 to seal the bag assembly 501. Once the bag assembly 501 is sealed it is sterilized, preferably by gamma-radiation or gas.
Operation of the Invention
Each of the culture chambers of the present invention operate in substantially similar manners. In each case, the chamber initially is empty and must be filled with suitable biological media containing the desired cells, tissues, or other biologicals. The media is conditioned before it is pumped into the growth chamber or bag assembly. Conditioned media has a particular pH and has been gassed to contain desired quantities of oxygen and/or other gases in solution. Preferably, the culture chamber is totally filled with media and has zero headspace.
Before the media is introduced into the growth chambers of the culture chambers 10, 100, 300 and 400, the space between the membrane support cylinder 28 and the membrane 37 is filled with media. Filling the space between the support cylinder 28 and the membrane 37 is accomplished by attaching a syringe to the effluent end of the culture chamber via a luer lock valve and drawing media into the tubing and through the surface pockets 36 to form a sheet of fluid between the cylinder 28 and the membrane 37. A pump can then be used to maintain the fluid pressure within the space between the cylinder 28 and the membrane 37, while the rest of the chamber is filled with media and cells via the fill ports in the walls of the chamber.
To assist in filling the culture chamber 10, 100, 200, 300, 400, or 500 with media, one or more gas removal means or fill means are incorporated into the chamber assembly. As media is pumped into the chamber, gas will be displaced and must have a release mechanism. There are a variety of means known in the art for releasing gas from media storage bags as the bags are filled with media. Similar gas releasing means will work in the present invention. A preferred embodiment of a gas releasing means is the gas removal port 425, shown in
The gas removal port 425 transverses both sides of the wall of the culture chamber and is sealingly attached to the wall of the chamber or bioreactor bag, be the chamber a hard walled tube such as sleeve 24 of culture chamber 10 as shown in
To release gas from the interior of the chamber, the needle of a syringe may be inserted through the septum and the gas released through the syringe. However, a more efficient means, shown in
The chamber will also typically have an introduction means or fill port 423 whereby media, cells, tissues, etc. can be introduced into the interior of the chamber. There are numerous means known in the art by which the chamber may be filled. In fact, the gas removal port 425 described above, can be used to introduce small quantities of media or cells into the chamber. Typically a syringe would be used to inject such materials into the chamber through the septum 485. However, for rapidly filling the chamber or introducing certain tissue or organoid materials, a larger port may be desired.
One embodiment of a rapid fill media port such as port 423 shown for embodiment 400 in
The stretched membrane 37 over the cylinder 28 permits development of a thin sheet of media to flow between the outer cylindrical surface of support cylinder 28 and membrane 37. Two-way perfusion through membrane 37 then permits nutrients to enter the bioreactive media inside the chamber, while waste products perfuse in the other direction into the sheet flow between cylinder 28 and membrane 37. Cells and larger sized molecules within the media in the chamber will not pass through the membrane 37 and concentrate within the chamber.
As shown in
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3682318 *||Jun 10, 1970||Aug 8, 1972||Amicon Corp||Tubular ultrafiltration membrane and support|
|US3722694 *||Jun 10, 1970||Mar 27, 1973||Romicon Inc||Filtration device|
|US4988623||Jun 30, 1988||Jan 29, 1991||The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration||Rotating bio-reactor cell culture apparatus|
|US5026650||Jun 30, 1988||Jun 25, 1991||The United States Of Amercia As Represented By The Administrator Of The National Aeronautics And Space Administration||Horizontally rotated cell culture system with a coaxial tubular oxygenator|
|US5104802||Jul 28, 1989||Apr 14, 1992||The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration||Hollow fiber clinostat for simulating microgravity in cell culture|
|US5153131||Dec 11, 1990||Oct 6, 1992||The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration||High aspect reactor vessel and method of use|
|US5153133||Apr 15, 1991||Oct 6, 1992||The United States Of America As Represented By The Administrator, National Aeronautics And Space Administration||Method for culturing mammalian cells in a horizontally rotated bioreactor|
|US5155034||Mar 2, 1989||Oct 13, 1992||Three-dimensional cell to tissue assembly process|
|US5155035||Jun 28, 1990||Oct 13, 1992||The United States Of America As Represented By The Administrator, Of The National Aeronautics And Space Administration||Method for culturing mammalian cells in a perfused bioreactor|
|US5449617||Sep 2, 1993||Sep 12, 1995||Heraeus Sepatech Gmbh||Culture vessel for cell cultures|
|US5576211 *||Apr 18, 1995||Nov 19, 1996||Heraeus Instruments Gmbh||Modular culture vessel for cell cultures|
|US5637477||Aug 16, 1994||Jun 10, 1997||The United States Of America As Respresented By The Administrator Of The National Aeronautics And Space Administration||Recombinant protein production and insect cell culture and process|
|US5686301||Sep 2, 1994||Nov 11, 1997||Heraeus Instruments Gmbh||Culture vessel for cell cultures|
|US5998202||Feb 25, 1998||Dec 7, 1999||Synthecon, Inc.||Multiple chamber diffusion vessel|
|US6022733||Dec 2, 1997||Feb 8, 2000||Tam; Yun K.||Simulated biological dissolution and absorption system|
|US6080581||Oct 12, 1999||Jun 27, 2000||Charles Daniel Anderson||Culture vessel for growing or culturing cells, cellular aggregates, tissues and organoids and methods for using same|
|US6107055 *||Jul 30, 1998||Aug 22, 2000||Roche Diagnostics Gmbh||Method and device for carrying out biochemical reactions|
|US6228607 *||Apr 22, 1996||May 8, 2001||Organogenesis Inc.||Bioreactor|
|1||FiberCell Hollow Fiber Cell Culture Systems, Dec. 1, 2004 printout, Internet at bellcoglass.com.|
|2||Hernaeus Instruments, Inc.; Genetic Engineering News, vol. 14, No. 12, Jun. 15, 1994.|
|3||Rai M. et al.; Expression systems for production of heterologous proteins, Current Science, vol. 80, No. 9, May 10, 2001, pp. 1121-1128.|
|4||Verma, R. et al.; Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems, Journal of Immunological Methods, 216, 1998, pp. 165-181.|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US8278101 *||Dec 3, 2010||Oct 2, 2012||Synthecon, Inc.||Stem cell bioprocessing and cell expansion|
|US20080138828 *||Dec 8, 2006||Jun 12, 2008||Marshall University Research Corporation||Microgravity bioreactor systems for production of bioactive compounds and biological macromolecules|
|US20110136226 *||Jun 9, 2011||Synthecon, Inc.||Stem cell bioprocessing and cell expansion|
|CN102996957A *||Dec 14, 2012||Mar 27, 2013||中国科学院力学研究所||Double-channel miniature rotary seal used for bioreactor|
|WO2012170878A2 *||Jun 8, 2012||Dec 13, 2012||Humacyte, Inc.||Apparatuses for tissue and organ production and storage|
|WO2012170878A3 *||Jun 8, 2012||May 8, 2014||Humacyte, Inc.||Apparatuses for tissue and organ production and storage|
|U.S. Classification||435/297.2, 435/298.1, 210/321.78, 210/321.89, 435/304.1, 210/321.6, 210/321.87, 435/295.3, 435/297.3, 435/299.1|
|International Classification||C12M1/04, C12M3/00, C12M1/10, C12N5/02, C12M1/14, C12P21/02, B01D63/00, C12M3/06, C12M1/00, C12M1/12, C07K14/47, C02F1/44, C12M3/04|
|Cooperative Classification||C07K14/47, C12M23/06, C12M25/02, C12N2510/02, C12M23/14, C12M29/06, C12P21/02, C12M29/10|
|European Classification||C12M29/10, C12M23/14, C12M29/06, C12M25/02, C12M23/06, C12P21/02, C07K14/47|
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